Noel

mBio. 2021 Dec 21;12(6):e0278621. doi: 10.1128/mBio.02786-21. Epub 2021 Dec 7.

ABSTRACT

The hospital-acquired pathogen Acinetobacter baumannii possesses a complex cell envelope that is key to its multidrug resistance and virulence. The bacterium, however, lacks many canonical enzymes that build the envelope in model organisms. Instead, A. baumannii contains a number of poorly annotated proteins that may allow alternative mechanisms of envelope biogenesis. We demonstrated previously that one of these unusual proteins, ElsL, is required for maintaining a characteristic short rod shape and for withstanding antibiotics that attack the septal cell wall. Curiously, ElsL is composed of a leaderless YkuD-family domain usually found in secreted, cell wall-modifying l,d-transpeptidases (LDTs). Here, we show that, rather than being an LDT, ElsL is actually a new class of cytoplasmic l,d-carboxypeptidase (LDC) that provides a critical step in cell wall recycling previously thought to be missing from A. baumannii. Absence of ElsL impairs cell wall integrity, morphology, and intrinsic resistance due to buildup of murein tetrapeptide precursors, toxicity of which is bypassed by preventing muropeptide recycling. Multiple pathways in the cell become sites of vulnerability when ElsL is inactivated, including l,d-cross-link formation, cell division, and outer membrane lipid homoeostasis, reflecting its pleiotropic influence on envelope physiology. We thus reveal a novel class of cell wall-recycling LDC critical to growth and homeostasis of A. baumannii and likely many other bacteria. IMPORTANCE To grow efficiently, resist antibiotics, and control the immune response, bacteria recycle parts of their cell wall. A key step in the typical recycling pathway is the reuse of cell wall peptides by an enzyme known as an l,d-carboxypeptidase (LDC). Acinetobacter baumannii, an "urgent-threat" pathogen causing drug-resistant sepsis in hospitals, was previously thought to lack this enzymatic activity due to absence of a known LDC homolog. Here, we show that A. baumannii possesses this activity in the form of an enzyme class not previously associated with cell wall recycling. Absence of this protein intoxicates and weakens the A. baumannii cell envelope in multiple ways due to the accumulation of dead-end intermediates. Several other organisms of importance to health and disease encode homologs of the A. baumannii enzyme. This work thus reveals an unappreciated mechanism of cell wall recycling, manipulation of which may contribute to enhanced treatments targeting the bacterial envelope.

PMID:34872350 | PMC:PMC8649774 | DOI:10.1128/mBio.02786-21

J Biol Chem. 2021 Dec 2:101464. doi: 10.1016/j.jbc.2021.101464. Online ahead of print.

ABSTRACT

Wall teichoic acid (WTA) polymers are covalently affixed to the Gram-positive bacterial cell wall and have important functions in cell elongation, cell morphology, biofilm formation, and β-lactam antibiotic resistance. The first committed step in WTA biosynthesis is catalyzed by the TagA glycosyltransferase (also called TarA), a peripheral membrane protein that produces the conserved linkage unit which joins WTA to the cell wall peptidoglycan. TagA contains a conserved GT26 core domain followed by a C-terminal polypeptide tail (CTT) that is important for catalysis and membrane binding. Here we report the crystal structure of the Thermoanaerobacter italicus TagA enzyme bound to UDP-ManNAc, revealing the molecular basis of substrate binding. Native mass spectrometry experiments support the model that only monomeric TagA is enzymatically active and that it is stabilized by membrane binding. Molecular dynamics simulations and enzyme activity measurements indicate that the CTT facilitates catalysis by encapsulating the UDP-ManNAc substrate, presenting three highly conserved arginine residues to the active site that are important for catalysis (R214, R221, and R224). From these data we present a mechanistic model of catalysis that ascribes functions for these residues. This work could facilitate the development of new antimicrobial compounds that disrupt WTA biosynthesis in pathogenic bacteria.

PMID:34864059 | DOI:10.1016/j.jbc.2021.101464

Bull Exp Biol Med. 2021 Dec;172(2):164-168. doi: 10.1007/s10517-021-05356-4. Epub 2021 Dec 2.

ABSTRACT

We studied the effect of bacterial wall peptidoglycan of 7 bacterial species on the competitive properties of human-associated microorganisms. Addition of peptidoglycan to the culture medium did not change the growth characteristics of the test cultures; however, an increase in the antagonism and hydrophobicity of Bifidobacterium sp. and Enterococcus sp. was observed, while the effect on enterobacteria was predominantly indifferent or inhibitory. The effect did not depend much on the source of peptidoglycan and was equally manifested on both indigenous and probiotic strains. The observed new property of peptidoglycan indicates its participation in the formation and functioning of microbiota. The obtained data on the regulation of the properties of microorganisms provide new possibilities for the correction and maintenance of host homeostasis through host-associated microbiota.

PMID:34855091 | DOI:10.1007/s10517-021-05356-4

mSystems. 2021 Dec 21;6(6):e0101721. doi: 10.1128/mSystems.01017-21. Epub 2021 Nov 30.

ABSTRACT

How cells control their shape and size is a fundamental question of biology. In most bacteria, cell shape is imposed by the peptidoglycan (PG) polymeric meshwork that surrounds the cell. Thus, bacterial cell morphogenesis results from the coordinated action of the proteins assembling and degrading the PG shell. Remarkably, during steady-state growth, most bacteria maintain a defined shape along generations, suggesting that error-proof mechanisms tightly control the process. In the rod-shaped model for the Gram-positive bacterium Bacillus subtilis, the average cell length varies as a function of the growth rate, but the cell diameter remains constant throughout the cell cycle and across growth conditions. Here, in an attempt to shed light on the cellular circuits controlling bacterial cell width, we developed a screen to identify genetic determinants of cell width in B. subtilis. Using high-content screening (HCS) fluorescence microscopy and semiautomated measurement of single-cell dimensions, we screened a library of ∼4,000 single knockout mutants. We identified 13 mutations significantly altering cell diameter, in genes that belong to several functional groups. In particular, our results indicate that metabolism plays a major role in cell width control in B. subtilis. IMPORTANCE Bacterial shape is primarily dictated by the external cell wall, a vital structure that, as such, is the target of countless antibiotics. Our understanding of how bacteria synthesize and maintain this structure is therefore a cardinal question for both basic and applied research. Bacteria usually multiply from generation to generation while maintaining their progenies with rigorously identical shapes. This implies that the bacterial cells constantly monitor and maintain a set of parameters to ensure this perpetuation. Here, our study uses a large-scale microscopy approach to identify at the whole-genome level, in a model bacterium, the genes involved in the control of one of the most tightly controlled cellular parameters, the cell width.

PMID:34846166 | PMC:PMC8631317 | DOI:10.1128/mSystems.01017-21

J Med Chem. 2021 Dec 9;64(23):17326-17345. doi: 10.1021/acs.jmedchem.1c01407. Epub 2021 Nov 30.

ABSTRACT

Herein, we report the design and synthesis of inhibitors of Mycobacterium tuberculosis (Mtb) phospho-MurNAc-pentapeptide translocase I (MurX), the first membrane-associated step of peptidoglycan synthesis, leveraging the privileged structure of the sansanmycin family of uridylpeptide natural products. A number of analogues bearing hydrophobic amide modifications to the pseudo-peptidic end of the natural product scaffold were generated that exhibited nanomolar inhibitory activity against Mtb MurX and potent activity against Mtb in vitro. We show that a lead analogue bearing an appended neopentylamide moiety possesses rapid antimycobacterial effects with a profile similar to the frontline tuberculosis drug isoniazid. This molecule was also capable of inhibiting Mtb growth in macrophages where mycobacteria reside in vivo and reduced mycobacterial burden in an in vivo zebrafish model of tuberculosis.

PMID:34845906 | DOI:10.1021/acs.jmedchem.1c01407

J Bacteriol. 2021 Nov 29:JB0049821. doi: 10.1128/JB.00498-21. Online ahead of print.

ABSTRACT

Gram-negative bacteria utilize glycerophospholipids (GPLs) as phospho-form donors to modify various surface structures. These modifications play important roles in bacterial fitness in diverse environments influencing cell motility, recognition by the host during infection, and antimicrobial resistance. A well-known example is the modification of the lipid A component of lipopolysaccharide by the phosphoethanolamine (pEtN) transferase EptA that utilizes phosphatidyethanoalmine (PE) as the phospho-form donor. Addition of pEtN to lipid A promotes resistance to cationic antimicrobial peptides (CAMPs), including the polymyxin antibiotics like colistin. A consequence of pEtN modification is the production of diacylglycerol (DAG) that must be recycled back into GPL synthesis via the diacylglycerol kinase A (DgkA). DgkA phosphorylates DAG forming phosphatidic acid, the precursor for GPL synthesis. Here we report that deletion of dgkA in polymyxin-resistant E. coli results in a severe reduction of pEtN modification and loss of antibiotic resistance. We demonstrate that inhibition of EptA is regulated post-transcriptionally and is not due to EptA degradation during DAG accumulation. We also show that the inhibition of lipid A modification by DAG is a conserved feature of different Gram-negative pEtN transferases. Altogether, our data suggests that inhibition of EptA activity during DAG accumulation likely prevents disruption of GPL synthesis helping to maintain cell envelope homeostasis.

PMID:34843376 | DOI:10.1128/JB.00498-21

J Immunol. 2021 Nov 19:ji2100486. doi: 10.4049/jimmunol.2100486. Online ahead of print.

ABSTRACT

The Stat signaling pathway plays important roles in mediating the secretions of a large number of cytokines and growth factors in vertebrates, which is generally triggered by the growth factor receptor, cytokine receptor, G protein coupled receptor, and receptor protein tyrosine kinase. In the current study, a platelet-derived growth factor receptor (defined as CgPDGFRβ) was identified from the Pacific oyster Crassostrea gigas, with a signal peptide, three Ig domains, a transmembrane domain, and an intracellular Ser/Thr/Tyr kinase domain. The two N-terminal Ig domains of CgPDGFRβ showed relatively higher binding activity to Gram-negative bacteria and LPS compared with Gram-positive bacteria and peptidoglycan. Upon binding bacteria, CgPDGFRβ in hemocytes formed a dimer and interacted with protein tyrosine kinase CgSrc to induce the phosphorylation of CgSrc at Tyr416. The activated CgSrc interacted with CgStat to induce the translocation of CgStat into the nucleus of hemocytes, which then promoted the expressions of Big defensin 1 (CgBigdef1), IL17-4 (CgIL17-4), and TNF (CgTNF1). These findings together demonstrated that the Src/Stat signaling was activated after the binding of CgPDGFRβ with bacteria to induce the expressions of CgBigdef1, CgIL17-4, and CgTNF1.

PMID:34799429 | DOI:10.4049/jimmunol.2100486

Org Biomol Chem. 2021 Oct 27;19(41):8906-8911. doi: 10.1039/d1ob01112j.

ABSTRACT

A major challenge in fluorescence imaging experiments, which are essential to determine protein activity, expression, and localization, is the penetration of small-molecule probes through the outer membrane permeability barrier of bacteria. Here, we describe a novel strategy for small-molecule probe-based fluorescence protein labeling and imaging in the Gram-negative bacterium Escherichia coli. We targeted a siderophore enterobactin biosynthetic enzyme EntE in E. coli. When coupled with an efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone, small-molecule probes were able to efficiently enter the cells, leading to the fluorescence labeling and imaging of overproduced EntE in E. coli. This study demonstrates that the combination of small-molecule probes with appropriate efflux pump inhibitors may substantially enhance their interaction with the target proteins in live bacteria.

PMID:34704577 | DOI:10.1039/d1ob01112j

Microbiol Spectr. 2021 Oct 31;9(2):e0052821. doi: 10.1128/Spectrum.00528-21. Epub 2021 Oct 20.

ABSTRACT

Staphylococcus aureus is an opportunistic pathogen that causes a wide range of infections. Due to the rapid evolution of antibiotic resistance that leads to treatment failure, it is important to understand the underlying mechanisms. Here, the cell wall structures of several laboratory vancomycin-intermediate S. aureus (VISA) strains were analyzed. Among the VISA strains were S. aureus VC40, which accumulated 79 mutations, including most importantly 2 exchanges in the histidine-kinase VraS, and developed full resistance against vancomycin (MIC, 64 μg/ml); a revertant S. aureus VC40R, which has an additional mutation in vraR (MIC, 4 μg/ml); and S. aureus VraS(VC40), in which the 2 vraS mutations were reconstituted into a susceptible background (MIC, 4 μg/ml). A ultraperformance liquid chromatography (UPLC) analysis showed that S. aureus VC40 had a significantly decreased cross-linking of the peptidoglycan. Both S. aureus VC40 and S. aureus VraS(VC40) displayed reduced autolysis and an altered autolysin profile in a zymogram. Most striking was the significant increase in d-alanine and N-acetyl-d-glucosamine (GlcNAc) substitution of the wall teichoic acids (WTAs) in S. aureus VC40. Nuclear magnetic resonance (NMR) analysis revealed that this strain had mostly β-glycosylated WTAs in contrast to the other strains, which showed only the α-glycosylation peak. Salt stress induced the incorporation of β-GlcNAc anomers and drastically increased the vancomycin MIC for S. aureus VC40R. In addition, β-glycosylated WTAs decreased the binding affinity of AtlA, the major autolysin of S. aureus, to the cell wall, compared with α-glycosylated WTAs. In conclusion, there is a novel connection between wall teichoic acids, autolysis, and vancomycin susceptibility in S. aureus. IMPORTANCE Infections with methicillin-resistant Staphylococcus aureus are commonly treated with vancomycin. This antibiotic inhibits cell wall biosynthesis by binding to the cell wall building block lipid II. We set out to characterize the mechanisms leading to decreased vancomycin susceptibility in a laboratory-generated strain, S. aureus VC40. This strain has an altered cell wall architecture with a thick cell wall with low cross-linking, which provides decoy binding sites for vancomycin. The low cross-linking, necessary for this resistance mechanism, decreases the stability of the cell wall against lytic enzymes, which separate the daughter cells. Protection against these enzymes is provided by another cell wall polymer, the teichoic acids, which contain an unusually high substitution with sugars in the β-conformation. By experimentally increasing the proportion of β-N-acetyl-d-glucosamine in a closely related isolate through the induction of salt stress, we could show that the β-conformation of the sugars plays a vital role in the resistance of S. aureus VC40.

PMID:34668723 | PMC:PMC8528128 | DOI:10.1128/Spectrum.00528-21

Front Microbiol. 2021 Sep 21;12:728989. doi: 10.3389/fmicb.2021.728989. eCollection 2021.

ABSTRACT

The healthy human epidermis provides physical protection and is impenetrable for pathogenic microbes. Nevertheless, commensal and pathogen bacteria such as Staphylococcus aureus are able to colonize the skin surface, which may subsequently lead to infection. To identify and characterize regulatory elements facilitating adaptation of S. aureus to the human skin environment we used ex vivo tissue explants and quantified S. aureus gene transcription during co-culture. This analysis provided evidence for a significant downregulation of the global virulence regulator agr upon initial contact with skin, regardless of the growth phase of S. aureus prior to co-culture. In contrast, the alternative sigma factor B (sigB) and the antimicrobial peptide-sensing system (graRS) were expressed during early colonization. Consistently, sigB target genes such as the clumping factor A (clfA) and fibrinogen and fibronectin binding protein A (fnbA) were strongly upregulated upon skin contact. At later timepoints of the adhesion process, wall teichoic acid (WTA) synthesis was induced. Besides the expression of adhesive molecules, transcription of molecules involved in immune evasion were increased during late colonization (staphylococcal complement inhibitor and staphylokinase). Similar to nasal colonization, enzymes involved in cell wall metabolism (sceD and atlA) were highly transcribed. Finally, we detected a strong expression of proteases from all three catalytic classes during the entire colonization process. Taken together, we here present an ex vivo skin colonization model that allows the detailed characterization of the bacterial adaptation to the skin environment.

PMID:34621255 | PMC:PMC8490888 | DOI:10.3389/fmicb.2021.728989

Adv Healthc Mater. 2021 Oct 6:e2101180. doi: 10.1002/adhm.202101180. Online ahead of print.

ABSTRACT

When searching for new antibiotics against Gram-negative bacterial infections, a better understanding of the permeability across the cell envelope and tools to discriminate high from low bacterial bioavailability compounds are urgently needed. Inspired by the phospholipid vesicle-based permeation assay (PVPA), which was designed to predict non-facilitated permeation across phospholipid membranes, outer membrane vesicles (OMVs) of Escherichia coli either enriched or deficient of porins are employed to coat filter supports for predicting drug uptake across the complex cell envelope. OMVs and the obtained in vitro model are structurally and functionally characterized using cryo-TEM, SEM, CLSM, SAXS and light scattering techniques. In vitro permeability, obtained from our membrane model for a set of nine antibiotics, correlates with reported in bacterio accumulation data and allows to discriminate high from low accumulating antibiotics. In contrast, the correlation of the same data set generated by liposome-based comparator membranes is poor. This better correlation of the OMV-derived membranes points to the importance of hydrophilic membrane components, such as lipopolysaccharides and porins, since those features are lacking in liposomal comparator membranes. This approach can offer in future a high throughput screening tool with high predictive capacity or can help to identify compound- and bacteria-specific passive uptake pathways. This article is protected by copyright. All rights reserved.

PMID:34614289 | DOI:10.1002/adhm.202101180

ACS Chem Biol. 2021 Nov 19;16(11):2527-2536. doi: 10.1021/acschembio.1c00604. Epub 2021 Oct 5.

ABSTRACT

Proteins from bacterial foes, antimicrobial peptides, and host immune proteins must navigate past a dense layer of bacterial surface biomacromolecules to reach the peptidoglycan (PG) layer of Gram-positive bacteria. A subclass of molecules (e.g., antibiotics with intracellular targets) also must permeate through the PG (in a molecular sieving manner) to reach the cytoplasmic membrane. Despite the biological and therapeutic importance of surface accessibility, systematic analyses in live bacterial cells have been lacking. We describe a live cell fluorescence assay that is robust, shows a high level of reproducibility, and reports on the permeability of molecules to and within the PG scaffold. Moreover, our study shows that teichoic acids impede the permeability of molecules of a wide range of sizes and chemical composition.

PMID:34609132 | DOI:10.1021/acschembio.1c00604

Bioorg Chem. 2021 Nov;116:105360. doi: 10.1016/j.bioorg.2021.105360. Epub 2021 Sep 15.

ABSTRACT

Proper recognition of invading pathogens and prompt initiation of host defense mechanisms are instrumental for the maintenance of organismal homeostasis. Nucleotide-binding oligomerization domain-containing (NOD)-like receptors (NLRs) serve as pathogen-recognition receptors that specifically recognize bacterial peptidoglycans. NOD2 detects muramyl dipeptide (MDP) through its carboxy-terminal leucine rich repeats (LRRs), which enables the activation of downstream inflammatory signaling. Synthesis of MDP conjugates based on solution phase chemistry have been previously reported. Our solid phase approach synthetically provides a facile approach for the conjugation of biological probes to MDP, with the advantage of minimal functional/protecting group manipulation, and reduction in the laborious process of intermediate purification and isolation. MDP conjugates that we generated using solid phase synthesis allow detection of NOD2 is cell lysates and NOD2 subcellular localization by immunofluorescence microscopy. MDP-PEG6-Cyanine5.5 conjugate selectively colocalized with WT NOD2 but not NOD2 variant found in Crohn's disease, which lacks carboxy-terminal end and cannot bind MDP. Overall, these data indicate that distinct solid phase-produced MDP conjugates can be used to examine biological properties of NOD2 and could potentially facilitate further development of NOD2 targeting agents.

PMID:34562676 | DOI:10.1016/j.bioorg.2021.105360

ACS Nano. 2021 Sep 17. doi: 10.1021/acsnano.1c04375. Online ahead of print.

ABSTRACT

Understanding how bacteria grow and divide requires insight into both the molecular-level dynamics of ultrastructure and the chemistry of the constituent components. Atomic force microscopy (AFM) can provide near molecular resolution images of biological systems but typically provides limited chemical information. Conversely, while super-resolution optical microscopy allows localization of particular molecules and chemistries, information on the molecular context is difficult to obtain. Here, we combine these approaches into STORMForce (stochastic optical reconstruction with atomic force microscopy) and the complementary SIMForce (structured illumination with atomic force microscopy), to map the synthesis of the bacterial cell wall structural macromolecule, peptidoglycan, during growth and division in the rod-shaped bacterium Bacillus subtilis. Using "clickable" d-amino acid incorporation, we fluorescently label and spatially localize a short and controlled period of peptidoglycan synthesis and correlate this information with high-resolution AFM of the resulting architecture. During division, septal synthesis occurs across its developing surface, suggesting a two-stage process with incorporation at the leading edge and with considerable in-filling behind. During growth, the elongation of the rod occurs through bands of synthesis, spaced by ∼300 nm, and corresponds to denser regions of the internal cell wall as revealed by AFM. Combining super-resolution optics and AFM can provide insights into the synthesis processes that produce the complex architectures of bacterial structural biopolymers.

PMID:34533301 | DOI:10.1021/acsnano.1c04375